PROJECT SUMMARY/ABSTRACT: Identifying host determinants governing HIV transcription and latency is critical to developing an HIV cure. Cell-surface glycosylation and lectin-glycan signaling play critical roles in the establishment of several immune responses and modulation of cell-cell and cell-pathogen interactions. The relevance of host glycosylation machinery to HIV latency is yet to be determined. We performed a pilot experiment involving the application of cutting-edge technologies to characterize the glycan structure profiles on the cell membranes of HIV latently- infected, productively-infected, and uninfected primary CD4+ T cells (obtained by infecting primary CD4+ T cells with a dual-fluorescence HIV reporter construct which enables the identification, quantification, and FACS-based purification of these cellular populations). Our pilot experiment strongly supports the hypothesis that latently-infected primary CD4+ T cells harbor a distinct glycomic profile, as compared to productively- infected or uninfected cells. Furthermore, we recently demonstrated that the human carbohydrate-binding protein galectin-9 (Gal-9) regulates HIV transcription and potently reactivates latent HIV in vitro and ex vivo. Gal-9 signals through cell-surface N-linked glycans in vitro, modulating key transcription initiation and chromatin remodeling factors that regulate HIV latency. Our data reveal that host glycan structures on the surface of infected cells may mediate signals that define the transcriptional state of HIV, and suggest that Gal- 9 and the host glycosylation machinery should be explored as foundations for novel strategies to cure HIV. Aim 1 of our proposal will utilize a primary cell-based model to rigorously determine if HIV latently-infected CD4+ cells exhibit a distinct glycomic fingerprint that can be exploited to identify and target HIV latency. We will infect primary CD4+ T cells isolated from 40 HIV-uninfected donors with a dual fluorescent reporter HIV construct allowing the differentiation and purification of latently-infected, productively-infected, and uninfected cells. We will identify glycan patterns associated with HIV latency by profiling the cell-surface glycan structures of each population using an advanced, high-density lectin microarray platform. In Aim 2, we will decipher the nature of glycan-mediated recognition in Gal-9-mediated reversal of HIV latency. First, cell-surface glycan patterns of purified HIV latently-infected primary CD4+ T cells will be correlated with the ability of Gal-9 to reverse HIV latency in vitro. Then, we will use enzymatic deglycosylation to examine the requirement of cell- surface N-linked and O-linked glycans in Gal-9-mediated viral reactivation ex vivo in primary CD4+ T cells isolated from HIV-infected ART-suppressed individuals. This study will allow us to define the opportunities by which glycan-based interventions can be harnessed to identify and eradicate HIV infection.